Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of microbial products. Ten distinct TLR proteins have been identified, which appear to be non-redundant with respect to the ligands they recognize. Different TLR agonists induce distinct patterns of gene expression in macrophages and dendritic cells, suggesting that the innate immune system is capable of mounting a specific response to the pathogen being recognized. The mechanistic basis for this specificity depends on differences in signal transduction pathways activated by the various TLR proteins. TLR proteins play important roles in host defense at the levels of both innate and adaptive immunity in humans and other mammals. These receptors also play critical roles in the development of effective vaccines that protect against infection. Indeed, many of the most potent vaccine adjuvants currently know are TLR agonists. Immunization with a defined peptide antigen has been shown to elicit a Thl- and Th2-type immune response, depending on whether the antigen was administered in the presence of a TLR4 or a TLR2 agonist, respectively. Thus, the capacity of different TLR proteins to evoke distinct patterns of gene expression helps to define the precise nature of the immune response evoked during infection or vaccination. The overall objective of these proposed studies is to characterize the signal transduction pathways that lead to the distinct patterns of gene expression induced by different TLR agonists. Our specific aims will: (1) Define regions within the intracellular signaling domains of TLR3 and TLR4 that are necessary for the activation of NF-kappaB, MAP kinases, and cytokine expression. (2) Define regions within the intracellular signaling domains of TLR3 and TLR4 that are necessary for the activation of PKC-d and PI-3K. (3) Assess the mechanism of TLR tyrosine phosphorylation and its functional consequences.